Materials and Methods Related to Sodium/Potassium Adenosine Triphosphatase and SRC

ABSTRACT

Described herein are methods to bind a compound to the SH2 domain of Src in a Src-expressing cell which includes contacting a compound comprising an amino acid compound to a Src-expressing cell; and binding a compound to the SH2 domain of Src.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/521,871 filed Aug. 2, 2012, now allowed, which is a nationalstage application filed under 35 USC §371 of international applicationPCT/US2011/021130 filed Jan. 13, 2011 which claims the benefit of U.S.Provisional Application No. 61/294,665 filed Jan. 13, 2010, the entiredisclosures of each of which are expressly incorporated herein byreference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant NumbersHL-36573 and HL-67963 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 13, 2011, isnamed 420_(—)52651_SEQ_LIST.txt, and is 1 kb in size.

FIELD OF THE INVENTION

This invention pertains to the field of biology, chemistry and medicine.The invention specifically pertains to ion transport proteins, smallpharmaco-active molecules, research tools, diagnostics, kits andtreatments related to cardiovascular diseases. Cardiotonic steroidantagonists and compositions affecting cholesterol-mediatedcardiovascular disease are within the field of the invention. Otherfields, such as physics and biochemistry also provide a framework forthe present invention.

BACKGROUND OF THE INVENTION

This invention is based in part on the elucidation of new structuralconformations and functions of the sodium/potassium adenosinetriphosphate synthase (Na/K ATPase), and especially elucidation of newbinding sites and interactions. The present invention providesapplications of surprising structural and functional relationshipsbetween Na/K ATPase and compounds which interact with Na/K ATPase. Theinvention provides solutions to chemically affecting not only the Na/KATPase interactions, but also regulators known to be upstream anddownstream.

Sodium and potassium transport activity across the cell membrane isintrinsically related to many cellular processes including metabolism,gene expression and cell growth. The Na/K-ATPase is highly expressed andrepresents one of the most fundamentally important proteins of animalphysiology. Moreover, the expression and the activity of Na/K-ATPase areimportant in regulation of the overall transport activity of a cell.This so-called pump/leak coupling exists in almost every mammalian cell.Because protein phosphorylation constitutes a pivotal mechanism by whichthe cellular processes are coordinated, the inventors postulate theexistence of a receptor mechanism that can couple the transmembranetransport activity of Na/K-ATPase to on/off of protein kinases.

It is known that a large number of Na/K-ATPases interact directly withSrc kinase in cultured cells as well as in vivo. The interactioninvolves at least two pairs of protein domains. Specifically, the secondcytosolic domain of al subunit (CD2) interacts with the Src SH2 and thenucleotide binding (N) domain associates with the Src kinase domain. Thelatter interaction keeps Src in an inactive state and binding ofcardiotonic steroids such as ouabain to the Na/K-ATPase/Src complexactivates the associated Src, resulting in the assembly and activationof various protein kinase cascades.

SUMMARY OF THE INVENTION

The present invention provides composition of matter comprising an aminoacid compound comprising at least ten consecutive amino acid residues ofthe sequence STNCV EGTAR GIVVY TGD [SEQ ID NO:1], or conservativesubstitutions of the at least ten consecutive amino acid residues,wherein the compound is capable of binding the SH2 domain of Src.

Preferred are those compositions, which further comprise atherapeutically acceptable excipient. Most preferred are those whereinthe amino acid compound comprises the sequence STNCV EGTAR GIVVY TGD[SEQ ID NO: 1].

Also preferred are those which bind to Src with an IC₅₀ selected fromthe group consisting of: less than about 90 nM; less than about 75 nM;less than about 50 nM; less than about 40 nM; less than about 30 nM,less than about 20 nM; less than about 10 nM; and less than 7 nM.

Also provided are nucleic acid compounds encoding a composition herein,as are vectors, and cells, particularly E. coli and mammalian cells,particularly tumor cells.

Also provided are monoclonal antibodies selective for a compositionherein, preferably those .which further comprise a detectable labelselected from the group consisting of: radioactive label; chemicallabel; fluorescent label; an antibody; and a protein. Preferred arethose compositions herein wherein the composition is capable ofaffecting a cellular process selected from the group consisting of:antagonizing a CTS-induced protein kinase cascade; upregulating a CTSinduced protein kinase cascade; Src inhibition; Src stimulation;Na/K-ATPase mimic; Na/K-ATPase competitive inhibitor; Lyn inhibition;Lyn stimulation; ouabain antagonism; ouabain stimulation; ERK1/2activation; ERK1/2 inhibition; membrane permeability by sodium ions;membrane permeability by potassium ions.

Also preferred are those compositions herein which further comprise atleast one additional therapeutic composition useful to a treat a diseaseselected from the group consisting of: cancer; vascular disease;cardiovascular disease; heart disease; prostate cancer; breast cancer;neuroblastoma; cardiac hypertrophy; tissue fibrosis; congestive heartfailure; ischemia/reperfusion injury; and Src-related diseases.

Also preferred are those compositions herein which further comprise asecond compound bound with the amino acid compound in a location otherthan SEQ ID NO: 1, wherein the second compound is selected from thegroup consisting of: chemotherapeutic drug; toxin; immunologicalresponse modifier; enzyme; and radioisotope.

In other broad embodiments, there are provided methods to bind acompound to the SH2 domain of in vitro, comprising contacting a compoundherein to at least one SH2 domain of Src. Preferred are those methods tobind a compound to the SH2 domain of Src in a Src-expressing cell,comprising contacting a compound of herein to at least oneSrc-expressing cell. More preferred are those methods wherein theSrc-expressing cell is a mammalian cell, particularly a monocyte, heartcell, liver cell, vascular cell; breast cell; prostate cell; kidneycell; muscle cell; blood cell; and brain cell. In vitro and in vivomethods are preferred, particularly animal models, particularly mouseand humans.

Also provided are methods of treating a Src-associated disease in amammal in need of such treatment, comprising administering a therapeuticcomposition herein. In particular, preferred are those methods whereinthe Src-associated disease is selected from the group consisting of:cancer; vascular disease; cardiovascular disease; heart disease;prostate cancer; breast cancer; neuroblastoma; cardiac hypertrophy;tissue fibrosis; congestive heart failure; and ischemia/reperfusioninjury. Methods for treating mammals, particularly those wherein themammal is human, are preferred.

Also provided are methods for treating cancer in a mammal in need ofsuch treatment, comprising administering a Src-inhibiting therapeuticcomposition herein.

Also provided are methods for vascular disease in a mammal in need ofsuch treatment, comprising administering a Src-inhibiting therapeuticcomposition herein.

Also provided are methods for cardiovascular disease in a mammal in needof such treatment, comprising administering a Src-inhibiting therapeuticcomposition herein.

Also provided are methods for heart disease in a mammal in need of suchtreatment, comprising administering a Src-inhibiting therapeuticcomposition herein.

Also provided are methods for treating prostate cancer in a mammal inneed of such treatment, comprising administering a Src-inhibitingtherapeutic composition herein.

Also provided are methods for treating breast cancer in a mammal in needof such treatment, comprising administering a Src-inhibiting therapeuticcomposition herein.

Also provided are methods for treating neuroblastoma in a mammal in needof such treatment, comprising administering a Src-inhibiting therapeuticcomposition herein.

Also provided are methods for treating cardiac hypertrophy in a mammalin need of such treatment, comprising administering a Src-inhibitingtherapeutic composition herein.

Also provided are methods for treating tissue fibrosis in a mammal inneed of such treatment, comprising administering a Src-inhibitingtherapeutic composition herein.

Also provided are methods for treating congestive heart failure in amammal in need of such treatment, comprising administering aSrc-stimulating therapeutic composition herein.

Also provided are methods for ischemia/reperfusion injury in a mammal inneed of such treatment, comprising administering a Src-stimulatingtherapeutic composition herein.

Also provided are methods for reducing increased basal Src activity in atumor cell, comprising administering a Src-inhibiting composition hereinto a Src-expressing tumor cell.

Also provided are methods for affecting FAK in a tumor cell comprisingadministering a Src-inhibiting composition herein to a Src-expressingtumor cell, particularly wherein the Src-expressing cell is a TCN cell.

Also provided are methods for reducing tumor cell migration in a tumorcell test model, comprising administering a Src-inhibiting compositionof claim 1 to a Src-expressing tumor cell.

Also provided are methods for killing cancer cells when the expressionof Na/K ATPase is reduced, comprising administering a Src-inhibitingcomposition herein to a Src-expressing tumor cell having reduced Na/KATPase expression.

Also provided are methods for inhibiting cell growth in a tumor cellline, comprising administering a Src-inhibiting composition herein to aSrc-expressing tumor cell line, particularly which further comprisescomparison of the ability of a composition herein to inhibit cell growthin a tumor cell line to a test compound's ability to inhibit cell growthin the same tumor cell line.

Also provided are methods for inhibiting prostate tumor cell growth in aprostate tumor cell line, comprising administering a Src-inhibitingcomposition herein to a Src-expressing prostate tumor cell line,particularly which further comprises comparison of the ability of acomposition of claim 1 inhibiting prostate tumor cell growth in aprostate tumor cell line to a test compound's ability to inhibit cellgrowth in the same prostate tumor cell line.

Also provided are methods for inhibiting breast tumor cell growth in abreast tumor cell line, comprising administering a Src-inhibitingcomposition herein to a Src-expressing breast tumor cell line,particularly which further comprises comparison of the ability of acomposition of claim 1 inhibiting prostate tumor cell growth in a breasttumor cell line to a test compound's ability to inhibit cell growth inthe same breast tumor cell line.

Also provided are methods for inhibiting neuroblastoma cell growth in aneuroblastoma tumor cell line, comprising administering a Src-inhibitingcomposition of claim 1 to a Src-expressing neuroblastoma tumor cellline, particularly which further comprises comparison of the ability ofa composition of claim 1 inhibiting neuroblastoma tumor cell growth in aprostate tumor cell line to a test compound's ability to inhibit cellgrowth in the same neuroblastoma tumor cell line.

Also provided are methods for screening at least one test composition todetermine whether the at least one composition affects Src, comprising:introducing a test composition comprising a modified amino acid compoundof STNCV EGTAR GIVVY TGD [SEQ ID NO: 1] to Src, wherein the modificationis at least one conservative amino acid substitution; and determiningwhether the test composition affects Src.

Preferred are methods as described above, wherein the affect is selectedfrom the group consisting of: Src binding; Src inhibition; Srcstimulation; Src function; Lyn binding; Lyn function; Lyn inhibition;ouabain antagonism; Na/K-ATPase function; ERK1/2 function; FAKinhibition; membrane permeability by sodium ions; membrane permeabilityby potassium ions. Also preferred are those methods as above, whereinintroducing a test composition is accomplished in vitro, in at least onemammalian cell, and/or in at least one tumor cell line. Also preferredare those methods as above, wherein determining whether the compositionaffects Src is measured by cell growth compared to control, by tumorgrowth compared to control, and/or by cell migration compared tocontrol, preferably in an animal model, most preferably in a NOD/SCIDmouse and/or a human.

Also provided are methods to screen for test compositions capable ofinhibiting binding between CD2 domain of Na/K ATPase and SH2 domain ofSrc, comprising the steps of: a. incubating test compositions withpurified Src for at least 5 and up to 45 minutes in a vessel; b.introducing Na/K ATPase to the vessel for at least 5 minutes and up to45 minutes; c. introducing ouabain to the vessel for at least 1 minuteand up to 45 minutes; d. introducing Mg2+/ATP to the vessel for at least0.5 minute and up to 45 minutes; e. conducting western blot analysis todetermine if Src activation was induced by ouabain. Preferred are suchmethods, wherein steps a. and b. are each 12-17 minutes, step c. is 2 to7 minutes, and step d. is at least one minute.

Also provided are methods to screen for test compositions capable ofinhibiting binding between CD2 domain of Na/K ATPase and SH2 domain ofSrc, comprising the steps of: a. contacting a test composition with aFRET pair of CD2 domain and SH2 domain; and b. identifying thosecompositions which abolish the FRET energy as capable of inhibiting theCD2/SH2 interaction. Preferred are those methods, wherein the CD2 islabeled with Cy3 and the SH2 is labeled with Cy5, particularly viaincubation in a 96 well plate, and most particularly, the FRET energy ismeasured using a spectra meter.

Various aspects of this invention will become apparent to those skilledin the art from the following detailed description of the preferredembodiment, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file may contain one or more drawings executedin color and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the U.S. Patent and Trademark Office upon request andpayment of the necessary fees.

FIGS. 1A-1B. The interaction between the Na/K-ATPase and Src.

FIG. 1A: GST pull-down analyses showing the concentration-dependentinteraction between Src and two domains of α1 subunit. The purificationof different constructs and the pull-down analyses were performed aspreviously described. Upper panel showed a representative Western blot.Lower panel showed the Commassie blue staining of GST, GST-CD2, andGST-ND1. n=3.

FIG. 1B: Modeling of Na/K-ATPase/Src interaction. Modeling of E1 and E2Na/K-ATPase was based on SERCA1a structure file 1SU4 and Na/K-ATPasestructure file 2zxe, and generated using SPDB View V3.7 program. The Adomain (N-terminus and CD2) in the α1 subunit was labeled in sky blue, Pdomain in green, N domain in red. The SH2 domain of Src was labeled inpurple, kinase domain in blue.

FIGS. 2A-2C. The E1 and E2-dependent regulation of Src by theNa/K-ATPase.

FIG. 2A and FIG. 2B: Regulation of the Na/K-ATPase-associated Src bychemical modifiers. The Na/K-ATPase was purified from pig kidney and thepurified Src was purchased from Upstate. To stabilize the Na/K-ATPase inthe E2P conformation, the purified Na/K-ATPase was treated by twodifferent fluoride compounds (BeF and AIF) as described, washed and thenincubated with Src. The ouabain-treated and control Na/K-ATPase/Srccomplex was used as positive and negative controls for the experiments,respectively. Similarly, the Na/K-ATPase was stabilized at the E1P stateby NEM/AMPPNP as described and then analyzed.

FIG. 2C, effects of chymotrypsin and trypsin digestion onNa/K-ATPase-mediated Src regulation. The purified Na/K-ATPase wassubjected to chymotrypsin or trypsin digestion as described. Thedigested enzyme was washed and assayed for Na/K-ATPase activity. Theenzyme preparations with less than 25% activity was incubated with Srcin 150 mM Na⁺ medium in the presence or absence of 10 μM ouabain. Thenon-digested enzyme was used as control. The activity of Src wasmeasured, and a representative Western blot was shown under eachexperimental condition. Quantitative data was presented as mean±S.E. ofat least 3 separate experiments. *, p<0.05; **, p<0.01.

FIGS. 3A-3G. Regulation of Na/K-ATPase-associated Src by different ions.

FIG. 3A, FIG. 3B and FIG. 3C: Effects of Na⁺ and K⁺ on theNa/K-ATPase-associated Src. The purified Na/K-ATPase was incubated withthe purified Src in the presence of different ligands for 15 min, andassayed for Tyr418 phosphorylation as in FIG. 2. Representative Westernblots are shown under each experimental condition. Quantitative datawere presented as mean±S.E. of at least three independent experiments.

FIG. 3D: Effects of extracellular K+ on Src in LLC-PK1 cells. Cells wereexposed to the normal medium (K⁺ 5 mM) or low K⁺ (K⁺ 1 mM) medium, andthen stained for Tyr(P)⁴¹⁸ Src as described. The scale bar represents 20μm.

FIG. 3E, FIG. 3F, and FIG. 3G: Cells were exposed to media containingdifferent concentrations of K⁺ or Na⁺, and then lysed. Cell lysates weresubjected to Western blot analyses of Src and ERKs as indicated.Quantitative data was presented as mean±S.E. of at three independentexperiments. *, p<0.05; **, p<0.01.

FIG. 4. CD2C2 peptide as a potent ouabain antagonist. CD2C2 peptide wasincubated with purified Src (the amount was indicated as molar ratio ofSrc/Cd2C2) for 15 minutes in PBS solution. Then purified Na/K-ATPasefrom pig kidney was added for another 15 minutes. After that, ouabainwas added to the reaction system for 5 minutes, followed by addition of2 mM Mg2+/ATP. The Src activity was determined by Western blot probingwith anti-phosphorylated Tyr418. A representative Western blot wasshown, and quantitative data was presented as mean±S.E. of at 3 separateexperiments. **, p<0.01.

FIG. 5. Establishment of YFP and YFP-CD2 stable cell lines. Theexpression of YFP and YFP-CD2 is shown in clone YFP Ctrl and CD2-2 celllines. The CD2 is from Pig Na/K ATPase (Swiss Prot ID P05024) aminoacids-152-288 (Cytoplasmic domain 2).

FIG. 6. Effects of expression of CD2 on cell growth. Control cell lines(LLC YFP and YFP Ctrl) and YFP-CD2 clones (CD2-1, CD2-2 and CD2-5).20,000 cells were platted in 12 well plate format and cell number werecounted using Trypan Blue staining with a Hemocytometer chamber for 24,48, 72 and 96 hrs. Data is plotted as shown above (combination of 3independent experiments, n=3).

FIGS. 7A-7B. CD2C2 Peptide as a Novel Ouabain Antagonist.

FIG. 7A, CD2C2 peptide attenuates the binding of CD2 to Src. CD2C2 wasadded to a mixture of Src and GST-CD2 at indicated concentrations. Srcin the pull-down products was analyzed by Western blot with anti-Srcantibody.

FIG. 7B, antagonizing ouabain-induced Src activation by CD2C2. Src waspreincubated with indicated amount of CD2C2 peptide for 15 min in PBS.Then Na/K-ATPase purified from pig kidney was added for another 15 min.1 μM ouabain was added to mixture for 5 min. Then Srcautophosphorylation was detected in the presence of 2 mM ATP/Mg²⁺. Arepresentative blot from three independent experiments is shown.

DETAILED DESCRIPTION OF THE INVENTION

The Na/K-ATPase undergoes E1/E2 conformational transition during an ionpumping cycle. A large number of cellular Na/K-ATPase also interactswith Src kinase via two pairs of domains. While the Na/K-ATPase actuatordomain binds the Src SH2 domain with a higher affinity than that betweenthe nucleotide binding domain and the Src kinase domain, the latterkeeps Src in an inactive state. The E1 Na/K-ATPase could bind both theSH2 and kinase domains simultaneously and that the transition from theE1 to E2 would release the kinase domain, resulting in the activation ofthe Na/K-ATPase-associated Src. Indeed, the inventors demonstrate thisconformation-dependent regulation of Src using purified enzymepreparations. Consistently, cellular conditions that increase theformation of the E2 Na/K-ATPase stimulate cellular Src activity and thedown-stream protein kinase cascades in live cells. Taken together, thesediscoveries now show a previously unrecognized signaling mechanism thatmay couple the change in conformational states of Na/K-ATPase toactivation/inhibition of cellular protein kinase cascades.

Based on the crystal structure of Na/K-ATPase, the CD2 constitutes apart of the actuator (A) domain. As depicted in FIG. 1A, the interactionbetween the A and SH2 domain exhibits higher affinity than that of Ndomain/kinase domain interaction. Moreover, the inventors' in vitrobinding data indicate that the Na/K-ATPase is likely to interact withboth the SH2 and kinase domains simultaneously in the absence ofouabain. During the pumping cycle, the Na/K-ATPase undergoes E1 to E2conformational transition where the N domain closes up and the A domainrotates to dock onto the N and P domains. Structure modeling suggeststhat the location of and the space between the A and N domains at the E2state are unlikely to allow the α1 subunit to bind both the SH2 and thekinase domains simultaneously (FIG. 1B).

Because the Src binding, even at the molar ratio of 1:1, exhibits nosignificant effect on Na/K-ATPase activity (FIG. 5), the inventors nowbelieve that the Na/K-ATPase could exert a conformation-dependentregulation of Src. Specifically, as illustrated in FIG. 1B, while the E1Na/K-ATPase inhibits Src, the E2 Na/K-ATPase would release the kinasedomain, resulting in the activation of Na/K-ATPase-associated Src. Therotation of A domain may be sufficient and necessary to push the kinasedomain off the moving N domain during the E1 to E2 transition of theNa/K-ATPase, resulting in the activation of Src.

The inventors utilized different chemical modifiers to stabilize theNa/K-ATPase in distinct conformational states and then assessed theconformation-dependent effect of Na/K-ATPase on Src. Fluoride compoundssuch as aluminum fluoride and beryllium fluoride, can interact withNa/K-ATPase as phosphate analogues, and stabilize the enzyme as well asother P-type ATPase in the E2P conformation. Thus, to demonstrate thestimulatory effect of E2 Na/K-ATPase on Src activity, the inventorsprepared AIF- and BeF-Na/K-ATPase and measured their effects on Src.

As depicted in FIG. 2A, like ouabain, treatment of the Na/K-ATPase witheither aluminum fluoride or beryllium fluoride was sufficient tostimulate the Na/K-ATPase-associated Src. On the other hand, when theNa/K-ATPase is stabilized in E1P by N-ethylmaleimide (NEM) and a slowlyhydrolysable ATP analogue adenyl-5′-yl imidodiphosphate (AMP-PNP), theNa/K-ATPase-associated Src is completely inhibited (FIG. 2B). As acontrol, the inventors also assessed whether these chemical modifiershave direct effect on Src. No significant effect was observed under thesame experimental conditions (data not shown).

The existence of E1 and E2 conformations of the Na/K-ATPase during thecatalytic cycle was first detected as distinct patterns of proteolyticcleavage in either Na⁺ or K⁺ medium. In the presence of Na⁺,chymotrypsin cleaves E1 Na/K-ATPase at Leu-266 in the A domain,producing an 83 kDa fragment. While the 83 kDa peptide retains theability to form phosphoenzyme intermediate (EP), the chymotrypsincleavage disrupts the coordinated movement of A and N domains, resultingin a complete inhibition of the ATPase activity. Thus, if thecoordinated movement of A and N domain is required for theconformation-dependent activation of the Na/K-ATPase-associated Src asdepicted in FIG. 1B, the inability of A domain to push off the Srckinase domain from the N domain would cause a complete inhibition ofSrc.

Indeed, as depicted in FIG. 2C, while Na⁺ (lane 1) and ouabain (lane 2)were able to stimulate the Na/K-ATPase-associated Src, they failed to doso after the Na/K-ATPase was digested by chymotrypsin in the presence ofNa⁺. To further confirm the importance of coordinated movement of A andN domain, the E2 Na/K-ATPase was digested by trypsin in the presence ofK⁺, washed, and then subjected to the same assay. Unlike chymotrypsin,trypsin cleaves E2 Na/K-ATPase at Arg-438 in the N domain, producing a48 kDa fragment that retains the capability of forming EP. However, likechymotrypsin digestion, disruption of the coordinated movement of A andN domains by trypsin was equally effective in inhibiting Na⁺ andouabain-induced activation of Src.

The above in vitro studies indicate that the Na/K-ATPase can turn Srcon/off through coordinated movements of A and N domains during E2/E1conformational transition. To verify the above findings and assess thephysiological relevance of this conformation-dependent regulation ofNa/K-ATPase-associated Src, the inventors incubated the purifiedNa/K-ATPase/Src complex in the presence of different ions that are knownto alter the formation of E1/E2 Na/K-ATPase. In comparison to choline,Na⁺ was more potent in converting E2 conformation to E1. Accordingly,addition of 150 mM Na⁺ to the reaction buffer caused a partialinhibition of Src (FIG. 3A). Moreover, in the presence of both Na⁺ andK⁺, the addition of ATP-Mg²⁺ would favor the formation of E1 Na/K-ATPaseand produced a further inhibition of Src (FIG. 3B). However, removal ofNa⁺ from this reaction buffer would increase the formation of E2Na/K-ATPase. Consistently, it stimulated the Na/K-ATPase-associated Src(FIG. 3B). Physiologically, the cellular Na/K-ATPase is exposed to about15 mM Na⁺ intracellularly and 5 mM K⁺ extracellularly. Under these ionicconditions, the inventors saw that most of Na/K-ATPase-associated Srcwas inactive. Lowering K⁺ from 5 mM to 1 mM produced a robuststimulation of Na/K-ATPase-associated Src (FIG. 3C). Taken together,these findings now show that the Na/K-ATPase-associated Src can beregulated by the substrate-induced conformational changes.

The above shows that Na/K-ATPase may control Src activity throughsubstrate-induced E1/E2 transitions. Because the balance of E1/E2Na/K-ATPase in live cells can be regulated by extracellular K⁺ andintracellular Na⁺, to further explore the physiological significance ofthis E1/E2-mediated Src regulation, the inventors measured Src activityin LLC-PK1 cells after the cells were exposed to differentconcentrations of extracellular K⁺. Lowering extracellular K⁺ would slowdown the dephosphorylation of E2P and accumulate E2 Na/K-ATPase in livecells.

As depicted in FIG. 3D, confocal imaging analyses showed that loweringextracellular K⁺ from 5 mM to 1 mM significantly increased cellularcontents of active Src in LLC-PK1 cells, consistent with the findingsdepicted in FIG. 3C. To verify these findings, the inventors alsoconducted Western blot analyses of cell lysates after the cells wereincubated in normal or low K⁺ medium. As shown in FIG. 3E, lowering K⁺from 5 mM to 1 mM, like addition of ouabain, not only stimulatedcellular Src activity, but also the down-stream kinase cascade of ERKs.

To verify that the Na/K-ATPase/Src complex is the receptor for lowK⁺-induced signal transduction in LLC-PK1 cells, the inventors repeatedthe above experiments in Na/K-ATPase-knock down PY-17 cells. PY-17 cellsare derived from LLC-PK1 cells that were transfected with plasmidsexpressing al-specific siRNA. In comparison to P-11 cells, PY-17 cellsexpress about 10% of α1 and have reduced number of Na/K-ATPase/Srccomplex.

As shown in FIG. 3F, lowering extracellular K⁺ from 5 to 1 mM failed toincrease cellular Src activity in PY-17 cells, indicating that theformation of Na/K-ATPase/Src complex is required for low K⁺ to stimulatecellular Src activity. This notion is further supported by the fact thatrescuing PY-17 cells by knock-in a rat al was sufficient to restore lowK⁺-induced Src activation.

To further confirm the ion-induced regulation of Na/K-ATPase/Srccomplex, the inventors also exposed the cells to low extracellular Na⁺.Lowering the extracellular Na⁺ would favor the formation of E1Na/K-ATPase in live cells. Consistently, the inventors observed thatdecreasing Na⁺ from 150 mM to 15 mM caused a further inhibition ofcellular Src activity as depicted in FIG. 3G.

These findings now show that the formation of Na/K-ATPase/Src complexallows not only extracellular ouabain, but also the pump substrates toregulate Src and Src effectors. Several unique properties of this novelcellular signaling mechanism are worthy of note.

The formation of this receptor complex involves a unique pair of domaininteractions. Specifically, the involvement of both A and N domains ofthe Na/K-ATPase in the interaction with Src kinase makes it possible forE2/E1 Na/K-ATPase to turn on/off the transducing activity of Src.

Also, the E1/E2 conformation-mediated signaling mechanism suggests thatthe Na/K-ATPase/Src complex can function as a receptor for bothextracellular and intracellular ligands of the Na/K-ATPase.

Moreover, the ion-mediated regulation of receptor Na/K-ATPase/Srccomplex could also serve as a major mechanism by which the cellcoordinates the pumping and leak activities across the plasma membranebecause the activation/inhibition of protein kinases is essential forregulating the activity and trafficking of many membrane transporters.For example, hypokalemia activates Src in intact animals. Moreover, thisactivation appears to be responsible for reduced surface expression ofROMK in renal epithelial cells, contributing to renal preservation of K⁺under the K⁺-restricted condition.

Finally, in addition to its substrates, many membrane and structuralproteins as well as lipids interact with the Na/K-ATPase and regulatethe formation of E1/E2 Na/K-ATPase. For example, the γ subunit increasesthe formation of E1-Na/K-ATPase. In addition, several naturallyoccurring mutants stabilize the pump in the E1 state. Thus, the normaloperation of Na/K-ATPase/Src receptor complex may provide cells a vitalmechanism to sensing both extracellular and intracellular cues, thuscoordinating various cellular processes.

These studies now show that the association between the Na/K ATPase andSrc involves two interacting pairs: one is between the ATPase secondcytosolic domain (CD2) and Src SH2 domain. The other is between theATPase nucleotide binding domain (N domain) and the Src kinase domain.While the simultaneous binding of both associating pairs keeps Srcinactive, ouabain stimulation can disrupt the latter pair and releaseSrc kinase from the N domain, triggering Src activation. The NaKtidederived from the N domain show potent inhibition of Src activity byinterfering with the kinase domain conformation.

The new peptide from the CD2 domain (amino acid sequence: STNCV EGTARGIVVY TGD [SEQ ID NO:1]), however, disrupts the interaction of theATPase CD2/Src SH2 domain, and abolishes ouabain-induced Srcstimulation. Thus, the peptides are more advantageous as specificouabain antagonists, with less interference with cellular Src kinaseactivity by themselves. However, a combination of the compositions wouldbe beneficial as well.

The present invention also provides assays for screening new chemicalsthat competitively inhibit the binding between CD2 and SH2, andantagonize ouabain stimulation.

EXAMPLES Example 1. CD2 Method

The inventors showed that the Na/K-ATPase binds Src via two domains. Theinteraction between the nucleotide-binding domain of Na/K-ATPase withSrc kinase domain inhibits Src activity whereas the interaction betweensecond cytosolic domain of Na/K-ATPase α subunit (CD2) and Src SH2SH3domain plays an important role in the formation of the receptorNa/K-ATPase/Src complex. To further test the significance of the latterinteraction, the inventors have generated stable cell lines thatexpressing the YFP-fused CD2. FIG. 5 shows the expression of YFP andYFP-CD2 in two stable cell lines the inventors have generated. Thesequence of CD2 is listed in the figure legend. Functionally, asdepicted in FIG. 6, expression of YFP-CD2 caused a significantinhibition of cell growth, indicating that CD2 plays an important rolein the formation of receptor Na/K-ATPase and thus in the regulation ofcell growth.

Structural modeling suggests that the binding motif is likely at theC-terminus of CD2. To support this notion, the inventors checked whetherthe peptides derived from C-terminus of CD2 can attenuate the bindingbetween CD2 and Src. Purified GST-CD2 was incubated with Src in theabsence and presence of peptides for 15 min. Then pull-down pellets wereanalyzed by Western blot with antibody against Src. As shown in FIG. 7A,peptide CD2C2, but not CD2C1, was able to compete with and block thebinding of GST-CD2 to Src SH2 domain. If CD2C2 peptide disrupts theSH2/CD2 interaction, the inventors expect that CD2C2 may abolishCTS-induced Src activation. To test that, the inventors preincubated Srcwith indicated amount of CD2 peptides for 15 min in PBS. Then purifiedpig kidney Na/K-ATPase was added for another 15 min. 1 μM ouabain wasadded to mixture for 5 min. Then Src autophosphorylation was detected inthe presence of 2 mM ATP/Mg²⁺. As shown in FIG. 7B, addition of CD2C2,but not CD2C1, to the mixture of purified Na/K-ATPase and Src, abolishedouabain-induced Src activation in a dose-dependent manner. Therefore, tosearch for new antagonists, the inventors will perform the followingfour sets of experiments.

First, the inventors will adapt a structure-based approach to look forCD2C2 mimetic. The inventors will synthesize smaller peptides andevaluate their potency as CTS antagonists. Structural information ofCD2C2 derivatives will be generated according to the availableNa/K-ATPase 3D structure (PDB ID: 2ZXE). Then the DOCK program will beused to search the NCI 3D structure database of more than 400,000 smallmolecules. Candidate compounds will be requested from the NCI. Theiractivities as CTS antagonists will be evaluated. Second, the inventorswill develop a high throughput assay using an ELISA format to searchchemical libraries for compounds that block ouabain-induced Srcactivation. Third, a FRET-based assay will be established to confirmwhether the antagonist (positive hits from the above study) targetsCD2/SH2. Briefly, CD2 and His-SH2SH3 will be expressed and purified. Thepurified proteins will be chemically labeled with a FRET fluorophorepair by targeting the available cysteine residues present in both CD2and SH2. To validate the FRET assay, the inventors will test the FRETefficiency between, for example, 488-CD2 and 568-SH2SH3, and assesswhether the CD2C2 peptide can reduce the FRED efficiency. CD2C1 will beused as a negative control. Needless to say, if the number of positivehits is limited, the inventors could simply test the effect of thesecompounds on CD2/SH2 interaction using the pull-down assay as in FIG.7A. Fourth, because a positive hit could also be a Src inhibitor ortarget to the Src kinase domain/N domain interaction or prevent thebinding of ouabain to the receptor Na/k-ATPase, the inventors will testthese possibilities with the compounds that do not affect theinteraction between the CD2 and SH2 domains.

Example 2

The inventors incubated the test chemicals with commercially availablepurified Src for 15 minutes in PBS solution. Then, purified Na/K ATPasefrom pig kidney was added for another 15 minutes. After that, ouabainwas added to the reaction system for five minutes, followed by additionof 2mM Mg2+/ATP. The Src activity was determined by Western blot probingwith anti-phosphorylated Tyr418. Ouabain treated Na/K ATPase/Src complexwas used as a positive control. The potential ouabain antagonist wasidentified if it inhibited Src activation under ouabain-inducingconditions. To further determine the specific inhibition of the bindingbetween CD2/SH2, a FRET pair of CD2 and SH2 was designed. CD2 waslabeled with Cy3, and SH2 was labeled with Cy5. Both peptides wereincubated in a 96 well plate, and the FRET energy transfer was measuredusing a spectra meter. The chemicals which abolished the FRET energytransfer were identified as ouabain antagonists/inhibitors of CD2/SH2interactions. These are cardiotonic steroid antagonists in general.

While the invention has been described with reference to various andpreferred embodiments, it should be understood by those skilled in theart that various changes may be made and equivalents may be substitutedfor elements thereof without departing from the essential scope of theinvention. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from the essential scope thereof.

Therefore, it is intended that the invention not be limited to theparticular embodiment disclosed herein contemplated for carrying outthis invention, but that the invention will include all embodimentsfalling within the scope of the claims.

The publication and other material used herein to illuminate theinvention or provide additional details respecting the practice of theinvention, are incorporated be reference herein, and for convenience areprovided in the following bibliography.

Citation of the any of the documents recited herein is not intended asan admission that any of the foregoing is pertinent prior art. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicant anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

We claim:
 1. A method to bind a compound to the SH2 domain of Src in aSrc-expressing cell comprising: a) contacting a compound comprising anamino acid compound consisting of the sequence STNCV EGTAR GIVVY TGD[SEQ ID NO: 1] to a Src-expressing cell; and b) binding a compound tothe SH2 domain of Src.
 2. The method of claim 1, wherein theSrc-expressing cell is a mammalian cell.
 3. The method of claim 1,wherein the mammalian cell is a monocyte.
 4. The method of claim 1,wherein the at least one mammalian cell is a cell selected from thegroup consisting of: heart cell; liver cell; vascular cell; breast cell;prostate cell; kidney cell; muscle cell; blood cell; and brain cell. 5.The method of claim 1, wherein the at least one mammalian cell iscultured in vitro.
 6. The method of claim 1, wherein the at least onemammalian cell is in an animal model.
 7. The method of claim 1, whereinthe at least one mammalian cell is human.
 8. A method of treating aSrc-associated disease in a mammal in need of such treatment comprising:a) administering to a mammal a therapeutic composition comprising anamino acid compound consisting of the sequence STNCV EGTAR GIVVY TGD[SEQ ID NO: 1]; and b) treating a Src-associated disease in the mammal.9. The method of claim 8, wherein the mammal is a human.
 10. The methodof claim 8, wherein the Src-associated disease is selected from thegroup consisting of: cancer; vascular disease; cardiovascular disease;heart disease; prostate cancer; breast cancer; neuroblastoma; cardiachypertrophy; tissue fibrosis; congestive heart failure; andischemia/reperfusion injury.
 11. A method of treating cancer in a mammalin need of such treatment comprising: a) administering to a mammal acomposition comprising an amino acid compound consisting of the sequenceSTNCV EGTAR GIVVY TGD [SEQ ID NO: 1]; and b) treating cancer in themammal.
 12. The method of claim 11, further comprising administering tothe mammal at least one additional therapeutic useful to treat cancer.13. The method of claim 11, wherein the composition inhibits growth ofthe cancer.
 14. The method of claim 11, wherein the composition reducesan increased basal Src activity in the cancer.
 15. The method of claim11, wherein the composition reduces migration of the cancer.
 16. Themethod of claim 11, wherein the composition causes cell death of thecancer.
 17. The method of claim 11, wherein the cancer is selected fromthe group consisting of: breast cancer; prostate cancer; andneuroblastoma.
 18. A method of treating a cardiovascular disease in amammal in need of such treatment comprising: a) administering to amammal a composition comprising an amino acid compound consisting of thesequence STNCV EGTAR GIVVY TGD [SEQ ID NO: 1]; and b) treating acardiovascular disease in the mammal.
 19. The method of claim 18,further comprising administering to the mammal at least one additionaltherapeutic useful to treat cardiovascular disease.
 20. The method ofclaim 18, wherein the cardiovascular diseases is selected from the groupconsisting of: cardiac hypertrophy; congestive heart failure; heartdisease; and ischemia/reperfusion; vascular disease; andatherosclerosis.
 21. A method of treating tissue fibrosis in a mammal inneed of such treatment comprising: a) administering to a mammal acomposition comprising an amino acid compound consisting of the sequenceSTNCV EGTAR GIVVY TGD [SEQ ID NO: 1]; and b) treating a cardiovasculardisease in the mammal.
 22. The method of claim 21, further comprisingadministering to the mammal at least one additional therapeutic usefulto treat tissue fibrosis.
 23. A method to screen for test compositionscapable of inhibiting binding between CD2 domain of Na/K ATPase and SH2domain of Src, comprising the steps of: a) incubating test compositionswith purified Src for at least 5 and up to 45 minutes in a vessel; b)introducing Na/KATPase to the vessel for at least 5 minutes and up to 45minutes; c) introducing ouabain to the vessel for at least 1 minute andup to 45 minutes; d) introducing Mg2+/ATP to the vessel for at least 0.5minute and up to 45 minutes; e) conducting western blot analysis todetermine if Src activation was induced by ouabain.
 24. The method ofclaim 23, wherein steps a) and b) are each about 12 minutes to about 17minutes, step c) is about 2 minute to about 7 minutes, and step d) is atleast one minute.
 25. A method to screen for test compositions capableof inhibiting binding between CD2 domain of Na/K ATPase and SH2 domainof Src, comprising the steps of: a) contacting a test composition with aFRET pair of CD2 domain and SH2 domain; and b) identifying thosecompositions which abolish the FRET energy as capable of inhibiting theCD2/SH2 interaction.
 26. The method of claim 25, wherein the CD2 islabeled with Cy3 and the SH2 is labeled with Cy5.